Cell fate patterning of the C. elegans vulva is an ideal system for the study of signal transduction mechanisms. EGF induces six equipotent vulval precursor cells (VPCs) to assume cell fates in a 3?-3?-2?-1?-2?-3? pattern with 99.8% accuracy. In the ?Morphogen Gradient? model for vulval patterning, distance from the EGF source dictates the fate of each VPC. In the ?Sequential Induction? model, the Ras?Raf?MEK?ERK MAP kinase cascade induces 1? fate, elicits DSL ligand production, and thus, via the LIN-12/Notch receptor, induces its two neighboring VPCs to become 2?. Because of the absence of key molecular details, these two models were debated for 16 years. We resolved this debate: to interpret the EGF gradient, presumptive 2? cells use Ras?RalGEF?Ral rather than the canonical Ras?Raf used in 1?s. Mutating RalGEF or Ral (a cousin of Ras) does not confer strong patterning defects, suggesting that sequential induction is the dominant patterning mechanism. These are clinically relevant molecules: Ras is the most mutated oncoprotein. Ras?RalGEF?Ral is thought to be equally important for oncogenesis as the canonical Ras?Raf. Our central hypothesis is that the Ras?RalGEF?Ral story justifies pursuing unexplored areas of VPC patterning because clinically important signals are found as positive and negative regulators. The objectives of this proposal are to unveil new facets of the molecular basis for the 1?/2? fate choice and its high reproducibility in vivo, exploiting the strengths of this system for dissection of signaling mechanisms. Our preliminary results support the feasibility of three aims, each with a hypothesis focused on distinct molecular mechanisms. Aim 1: Our CRISPR-generated activating mutation in endogenous Rap1 (Ras proximal) induced ectopic 1? cells. Lack of Rap1 reduces 1? cell induction. We will test the hypothesis that Rap1 is the nexus of two opposed regulatory inputs that promote and repress ERK activation in presumptive 1? vs 2? cells, respectively. Aim 2: We identified a novel Ral effector, GCK-2/MAP4 kinase, that possibly signals via p38 MAP kinase to promote 2? fate. We will test the hypothesis that Ral?GCK-2 triggers a p38 cascade. We will also test whether Ral?GCK-2 signal regulates CCCH RNA binding proteins to stabilize 3?UTRs of 2?-promoting genes. Aim 3: MIG-15/MAP4K, the sole paralog of GCK-2/MAP4K, paradoxically inhibits 2? fate. We will test the hypothesis that MIG-15 triggers a JNK MAP kinase cascade, which may be the missing signal that represses the Notch receptor in presumptive 1 cells. MIG-15 defines a novel class of vulval-specific Notch repressors. We will make use of the results of a screen we have completed for MIG-15-like targets to identify new players. For all three aims we will use CRISPR-engineered endogenous fluorescent reporters to deconvolute specific signals embedded within the vulval signaling network. Successful completion of these aims will define components and organizational principles of the vulval signaling network that leads to the exceptional fidelity of patterning, which are likely to apply both broadly and specifically to other developmental systems.